Difference between revisions of "Team:UPF CRG Barcelona/Overexpression"

Enhancing E. Coli beta-oxidation

In order to obtain a system with an enhanced long chain fatty acid (LCFA) uptake and degradation we first considered the overexpression of endogenous E. coli fatty acid (FA) degradation gene family.

LCFAs are essential membrane components and constitute important members of signalling pathways. E. coli can use FAs with various chain lengths as sole source of carbon and energy [1]. After their uptake, these FAs can either be degraded via the beta-oxidation pathway or used as precursors for membrane phospholipid biosynthesis. The degradation pathway is catalysed by the enzymes encoded by the fad regulon, which includes fadL, fadD, fadE, fadA and fadB[2].

fadL and fadD are responsible for LCFA uptake and activation for their previous metabolism. FadL is an outer membrane homodimeric channel involved in the transport of external LCFA to the periplasm [3]. To metabolize FAs, they must be activated to acyl-CoA esters. This step is performed through FadD, an inner membrane-associated acyl-CoA synthase [4]. Finally, the complete oxidation of activated LCFA is catalysed by the other three fatty-acid degradation gene family: fadE, fadA and fadB.

Figure 1 | Scheme representing fatty acid intracellular uptake and degradation Both unsaturated and saturated fatty acids can enter inside the cell through FadL, an outer membrane channel. Once in the periplasm, FadD allows the transport of this fatty acids inside the cytosol using a system coupled to acyl-coA ester formation. Finally, the beta-oxidation process starts under catalysis of FadE and FadH enzymes, followed by FadA and FadB, both of them forming a tetrameric complex which is responsible of hydration, oxidation and thiolytic cleavage.

Fatty acid degradation and biosynthesis pathways must have a very dynamic expression system according to the availability of FAs in the environment to maintain cell functionality [5]. Thus, fad genes expression depends directly on the presence of internal LCFA bound to acyl-CoA.

FadR protein is the main transcriptional regulator of fad genes and is the key element regulating this process [6, 7]. It is involved in the negative regulation of LCFA degradation; in the absence of FAs it is constitutively bound to the fad genes promoter, repressing its transcription. In the presence of LCFA in the medium conversion to acyl-coA esters occurs. Then, LCFA-CoA bind to FadR, causing its release from the promoters and allowing the transcription of fad genes [8].

Figure 2 | The pfadBA promoter is constitutively repressed by FadR protein. LCFA enter the cell through the outer membrane receptor fadL, then, they get bound to acety-CoA. LCFA-acetyl-CoA is able to bind to FadR which allows it to be released from the promoter making the later active

FadL and FadD play a key role in the regulation of this pathway, as they are the main responsible of the uptake of LCFA and its proper conversion to acyl-coA ester in the cytosol. We hypothesized that overexpressing FadL and FadD, in presence of extracellular LCFA, would produce an scenario in which LCFA-CoA would repress FadR, resulting in an enhanced expression of the rest of the fad genes. Thus, FadL and FadD were targeted in order to develop a system with an increased LCFA degradation metabolism.

To do so, we expressed the sequences of either FadD or FadL downstream the TetR repressible promoter (BBa_R0040). Thus, allowing to tune the protein expression levels with the addition of Anhydrotetracycline (ATC).

Figure 3 | Schematic representation of the fad gene construct. Scheme representing the inducible expression system of the fad genes. A double terminator (BBa_B0014), a weak rbs (BBa_B0032), the inducible promoter (BBa_R0040) and the target gene were coupled in this biobrick.

Growth assays were performed in order to study the metabolic burden caused by the expression of these constructs. Moreover, in order to analyse the functionality of these genetic units, palmitic acid in the medium was quantified using cupric-acetate. Therefore, an increase of LCFA uptake when overexpressing these two fad genes could be measured.

The main approach of our project relies on an effective plasmid overexpression system. However, plasmidic presence within a live therapeutic in the intestine could deem not so effective, by posing a threat to the patients’ health due to the spread of antibiotic resistance to the natural gut ecosystem by plasmid transmission from our probiotic. In order to solve this, we approached overexpression of the mentioned genes from a different perspective; by directly modifying the E. coli genome as explained in the integration section.

References:

[1] Iram, S. H., & Cronan, J. E. (2006). The β-oxidation systems of Escherichia coli and Salmonella enterica are not functionally equivalent. Journal of bacteriology, 188(2), 599-608.

[2] Fujita, Yasutaro, Hiroshi Matsuoka, and Kazutake Hirooka. "Regulation of fatty acid metabolism in bacteria." Molecular microbiology 66.4 (2007): 829-839.

[3] Lepore BW, Indic M, Pham H, Hearn EM, Patel DR, van den Berg B: Ligand-gated diffusion across the bacterial outer membrane. Proc Natl Acad Sci U S A 2011, 108:10121–10126.

[4] Weimar JD, DiRusso CC, Delio R, Black PN: Functional role of fatty acyl-coenzyme A synthetase in the transmembrane movement and activation of exogenous long-chain fatty acids. Amino acid residues within the ATP/AMP signature motif of Escherichia coli FadD are required for enzyme activity and fatty acid transport. J Biol Chem 2002, 277:29369–29376.

[5] Janßen, H. J., & Steinbüchel, A. (2014). Fatty acid synthesis in Escherichia coli and its applications towards the production of fatty acid based biofuels. Biotechnology for biofuels, 7(1), 7.

[6] Henry MF, Cronan JE Jr: Escherichia coli transcription factor that both activates fatty acid synthesis and represses fatty acid degradation. J Mol Biol 1991, 222:843–849.

[7] Henry MF, Cronan JE Jr: A new mechanism of transcriptional regulation: release of an activator triggered by small molecule binding. Cell 1992, 70:671–679.

[8] Feng Y, Cronan JE Jr: Crosstalk of Escherichia coli FadR with global regulators in expression of fatty acid transport genes. PLoS One 2012, 7:e46275.